Anti-TIGIT antibody pharmaceutical composition and application thereof
A composition and drug technology, applied in the direction of drug combination, antibody, drug delivery, etc., can solve problems such as TIGIT antibody application
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Embodiment 1
[0175] Embodiment 1, the preparation of anti-TIGIT antibody
[0176] 1. Preparation of Antigen and Antibody
[0177] 1.1 Protein design and expression
[0178] Using human TIGIT protein (Uniprot number: Q495A1) as the template of TIGIT of the present disclosure, design the amino acid sequence of the antigen and detection protein of the present disclosure, optionally fuse different tags on the basis of TIGIT protein, and clone them respectively on the pHr vector ( Self-produced) or pXC-17.4 vector (LONZA), transiently expressed in 293 cells or stably expressed and purified in CHO cells, to obtain the protein encoding the disclosed antigen and detection. The following TIGIT antigens refer to human TIGIT unless otherwise specified.
[0179] Fusion protein of TIGIT extracellular region and mouse IgG2aFc fragment: TIGIT-mFc, for immunization and detection
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[0182] Note: The underlined part is the signal peptide, and the italic part is mFc.
[0183] Fusio...
Embodiment 2
[0435] Embodiment 2, anti-TIGIT antibody activity test experiment
[0436] One: ELISA experiment of TIGIT antibody binding to human TIGIT protein
[0437]The binding ability of the anti-TIGIT antibody was detected by ELISA experiment between the antibody and human TIGIT protein. The TIGIT fusion protein with Fc or mFc tag is immobilized in a 96-well microtiter plate by binding to the anti-Fc or mFc antibody coated in the microtiter plate, and the strength of the signal after the antibody is added is used to judge the antibody and TIGIT The positive control molecules are 22G2-H3Q and 10A7 hIgG4 (wherein the light and heavy chain variable region sequence of 10A7 hIgG4 is from SEQ ID NO: 21 and 22 in US20130251720A1, and its light and heavy chain variable region is connected to form the light and heavy chain of the full-length antibody The constant regions are respectively SEQ ID NO: 79 and 78 of the present application) The specific experimental method is as follows:
[0438] ...
Embodiment 3
[0490] The mensuration of embodiment 3.TIGIT antibody Tm value
[0491] Prepare 10mM disodium hydrogen phosphate-citric acid (PB-CA) buffer solutions with different pH values (see 1) to 6) below), and prepare antibody preparations with an anti-TIGIT antibody concentration of 1 mg / ml:
[0492] 1) 10mM disodium hydrogen phosphate-citric acid, pH4.5
[0493] 2) 10mM disodium hydrogen phosphate-citric acid, pH5.0
[0494] 3) 10mM disodium hydrogen phosphate-citric acid, pH5.5
[0495] 4) 10mM disodium hydrogen phosphate-citric acid, pH6.0
[0496] 5) 10mM disodium hydrogen phosphate-citric acid, pH6.5
[0497] 6) 10mM disodium hydrogen phosphate-citric acid, pH7.0
[0498]The thermal stability of anti-TIGIT antibody in each group of preparations was determined by differential scanning calorimetry (DSC). The test results are shown in Table 20, and the results show that the anti-TIGIT antibody is relatively stable at pH 5.0-7.0.
[0499] Table 20. Tm values of different pH...
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